Adult testicular cells,neural stem cells (both fetal and adult),bone marrow cells, erbBeta2-induced mouse mammary tumor cells and human embryonal cancer cells, were mixed with limiting dilutions of dispersed mammary epithelial cells and injected into epithelium-divested mammary fat pads. This approach allowed for the growth of mammary tissue from the injected cells.The tissue microenvironment directs stem/progenitor cell behavior. Cancer cells are also influenced by the microenvironment. It has been shown that, when placed into blastocysts, cancer cells respond to embryonic cues and differentiate according to the tissue type encountered during ontological development. Previously, we demonstrated that the mouse mammary gland was capable of redirecting adult mouse testicular and neural stem/progenitor cells toward a mammary epithelial cell fate during gland regeneration. Here, we report that human embryonal carcinoma cells proliferate and produce differentiated mammary epithelial cell progeny when mixed with mouse mammary epithelial cells and inoculated into the epithelium-free mammary fat pads of athymic nude mice. Fluorescent In Situ Hybridization (FISH) confirmed the presence of human cell progeny in the mammary outgrowths for human centromeric deoxyribonucleic acid (DNA),and immunochemistry for human specific breast epithelial cytokeratins and human specific milk proteins in impregnated transplant hosts. It was found that the number of human cells increased by 66 to 660 fold during mammary epithelial growth and expansion as determined by human cytokeratin expression. All features found in primary outgrowths were recapitulated in the secondary outgrowths from chimeric implants. These results demonstrate that human embryonal carcinoma-derived progeny interact with mouse mammary cells during mammary gland regeneration and are directed to differentiate into cells that exhibit diverse mammary epithelial cell phenotypes. This is the first demonstration that human cells are capable of recognizing the signals generated by the mouse mammary gland microenvironment present during gland regeneration in vivo.The microenvironment of the mammary gland has been shown to exert a deterministic control over cells from different normal organs during murine mammary gland regeneration in transplantation studies. When MMTV-neu induced tumor cells were mixed with normal mammary epithelial cells (MECs) in a dilution series and inoculated into epithelium-free mammary fat pads, they were redirected to non-carcinogenic cell fates by interaction with untransformed mammary epithelial cells during regenerative growth. In the presence of non-transformed MECs (50 to 1), tumor cells interacted with MECs to generate functional chimeric outgrowths. When injected alone, tumor cells invariably produced tumors. Here, the normal microenvironment redirects MMTV-neu-transformed tumorigenic cells to participate in the regeneration of a normal, functional mammary gland. In addition, the redirected tumor cells demonstrate the capacity to differentiate into normal mammary cell types including luminal, myoepithelial and secretory. The results indicate that signals emanating from a normal mammary microenvironment, comprised of stromal, epithelial and host-mediated signals combine to suppress the cancer phenotype during glandular regeneration. Clarification of these signals offers improved therapeutic possibilities for the control of mammary cancer growth.The mouse mammary epithelial cell hierarchy contains both multipotent stem cell as well as lineage limited duct and lobular progenitor cell functions. The latter, also termed parity-identified mammary epithelial cells (PI-MEC)--are marked by beta-galactosidase expression following pregnancy and involution in whet acidic protein promoter-driven Cre (WAP-Cre)/Rosa26-flox-stop-flox-lacZ (WC/R26) mice and are the targets of tumorigenic transformation in MMTV-neu transgenic mice. Here we demonstrate that an epithelial population distinct from PI-MEC is transformed during WAP-Int3 tumorigenesis. As expected, WAP-Int3/WC/R26 triple-transgenic mice failed to undergo secretory alveolar development failed to lactate and developed mammary tumors. Following pregnancy and involution, beta galactosidase-positive mammary epithelial cells were found in the normal mammary tissue, but the resulting mammary tumors were all beta galactosidase negative. WAP-Int3/WC/R26 mammary glands contained ample estrogen receptor alpha-positive (ER-alpha-positive) mammary epithelial cells (MEC), but only rare (&lt;1%) progesterone receptor-positive (PR-positive) and RANKL-positive cells. In addition, dissociated MEC from WAP-Int3/WC/R26 glands failed to regenerate a mammary tree upon transplantation into a cleared fat-pad of a immune-compromised nude recipient mouse. However, when mixed with normal MEC, PI-MEC from WAP-Int3/WC/R26 mice contributed progeny to the resulting functional outgrowth. The WAP-Int3/WC/R26 derived PI-MEC displayed all of the properties of fully functional lobular progenitors including giving rise to ER-alpha-positive, PR-positive, smooth muscle actin-positive (SMA-positive), and RANKL-positive epithelial progeny. These results demonstrate that WAP-Int3 has no oncogenic effect upon PI-MEC and that the expansion of functional lobular progenitors is required for secretory alveolar development and lactation. Furthermore, lobular progenitor function is ultimately controlled by signals within its microenvironment.The microenvironment of the mammary gland has been shown to exert a deterministic control over cells from different normal organs during murine mammary gland regeneration in transplantation studies. When MMTV-neu induced tumor cells were mixed with normal mammary epithelial cells (MECs) in a dilution series and inoculated into epithelium-free mammary fat pads, they were redirected to non-carcinogenic cell fates by interaction with untransformed mammary epithelial cells during regenerative growth. In the presence of non-transformed MECs (50 to 1),tumor cells interacted with MECs to generate functional chimeric outgrowths. When injected alone, tumor cells invariably produced tumors. Here, the normal microenvironment redirects MMTV-neu transformed tumorigenic cells to participate in the regeneration of a normal, functional mammary gland. In addition, the redirected tumor cells demonstrate the capacity to differentiate into normal mammary cell types including luminal, myoepithelial and secretory. The results indicate that signals emanating from a normal mammary microenvironment, comprised of stromal, epithelial and host mediated signals combine to suppress the cancer phenotype during glandular regeneration. Clarification of these signals offers improved therapeutic possibilities for the control of mammary cancer growth. We are currently testing the hypothesis that non-cellular extracts from normal mammary tissue can reprogram non-mammary and cancer cell in the epithelium-divested mammary fat pad.